The use and abuse of tobacco remains one of the major causes of a myriad of health problems. It is well established that nicotine is the most important psychoactve substance in tobacco smoke and that smokers modify their use of tobacco to regulate nicotine intake. Smoking also appears to be regulated by genetic factors in humans. The studies outlined in this proposal will continue to use genetic strategies to attempt to explain variability in response to a first dose of nicotine (initial sensitivity). We have shown that inbred mouse strains differ in initial sensitivity to nicotine and that 35-40% of this variability correlates with variability in nicotinic receptor numbers; strains that have higher numbers of L[3H]- nicotine binding sites are more sensitive to several low dose effects of nicotine whereas strains that have high numbers of alpha[125I]- bungarotoxin binding sites are more sensitive to seizures that are evoked by high doses of nicotine. The studies outlined here will attempt to determine whether differences in brain nicotinic receptor function and desensitization also contribute to strain differences in response to nicotine. Initial studies will focus on two recently developed-essays (dopamine release from striatal synaptosomes and (86)Rb+ efflux from midbrain synaptosomes). Since the dopamine release process is very sensitive to inhibition by neuronal bungarotoxin it seems likely that the striatal nicotinic receptor that mediates this process contains an alpha-3 subunit. In contrast, the (86)Rb+ efflux from midbrain seems to be regulated by an alpha-4-containing receptor. Additional assays will also be developed and characterized by assessing the rank order of potency and efficacy of eight agonists and selected antagonists, especially neuronal bungarotoxin and alpha-bungarotoxin. Data obtained from these studies will be compared with results obtained by others using known nicotinic receptor combinations expressed in frog oocytes to provide a tentative assignment of receptor type. Subsequently, the kinetics of receptor desensitization and resensitization will be determined for each of the functional assays in each of the brain regions; these properties will be compared using brain tissue from mouse strains that differ in sensitivity to nicotine. We have also shown that chronic nicotine treatment results in tolerance to nicotine; some mouse strains develop tolerance at lower infusion doses than are required to elicit tolerance in others. Chronic nicotine infusion also evokes what has come to be known as a paradoxical up-regulation of brain nicotinic receptors. This change in receptor numbers parallels tolerance development in some mouse strains, but not in others. We have speculated that the unexpected up-regulation occurs because chronic nicotine treatment serves to desensitize or perhaps even inactivate brain nicotinic receptors, and have recently obtained evidence that suggests that this may be true, but brain regions may respond differently. The proposed studies will utilize the functional assays that we have developed and intend to develop to determine whether inbred mouse strains that differ in proclivity for developing tolerance do so because of differences in effects of chronic nicotine treatment on receptor function. The effects of chronic nicotine treatment will be compared with the effects of chronic mecamylamine treatment because we have recently observed that chronic antagonist treatment also elicits up-regulation of brain nicotinic receptor numbers. The results of these studies should provide much-needed insight into the role of nicotinic receptors in regulating response to nicotine, and may provide explanations for genetically-determined differences in response to nicotine that may be useful in explaining individual differences among humans in smoking behaviors.